Dear Brian,

I think your thought that the protein is coming out of solution is the correct one as sometimes refolding proteins on a column does not work. I think the only way to solve this problem is by trial and error. For two different proteins, when I worked at NIEHS, we looked first, using different buffer systems provided in a kit, to see what was the solubility after a simple dilution of the protein that was initially solubilized in urea. Or we solubilized the proteins directly from inclusion bodies using high pressure in a Barafold apparatus. Once we knew the best buffer system, we used this to try refolding on a column. The refolding on the column worked well for one protein but not for the other.

I used about 3 column volumes of 6M urea in your buffer of choice, followed by 3 column volumes of 4,2, 1 and 0.5 M urea in a buffer of choice. Then I typically eluted the protein with buffer containing 0.5M imidazlole, and buffer of choice. I typically did not wash with no urea buffer. Maybe someone else can comment on the lowest urea concentration that can be used for refolding. If the urea concentration needs to be lower than 0.5M, you can try washing with buffer without Urea. The other thing I had in the original denaturing buffer was 5mM TCEP. The pH needs to be adjusted on the Buffer if TCEP is added. Or you can make a stock solution of TCEP and pH that.

Also, I kept the unfolded protein in the urea/TCEP solution overnight in the cold and then centrifuged the solution, before applying to the column.

Dear Brian,

I think your thought that the protein is coming out of solution is the correct one as sometimes refolding proteins on a column does not work. I think the only way to solve this problem is by trial and error. For two different proteins, when I worked at NIEHS, we looked first, using different buffer systems provided in a kit, to see what was the solubility after a simple dilution of the protein that was initially solubilized in urea. Or we solubilized the proteins directly from inclusion bodies using high pressure in a Barafold apparatus. Once we knew the best buffer system, we used this to try refolding on a column. The refolding on the column worked well for one protein but not for the other.

I used about 3 column volumes of 6M urea in your buffer of choice, followed by 3 column volumes of 4,2, 1 and 0.5 M urea in a buffer of choice. Then I typically eluted the protein with buffer containing 0.5M imidazlole, and buffer of choice. I typically did not wash with no urea buffer. Maybe someone else can comment on the lowest urea concentration that can be used for refolding. If the urea concentration needs to be lower than 0.5M, you can try washing with buffer without Urea. The other thing I had in the original denaturing buffer was 5mM TCEP. The pH needs to be adjusted on the Buffer if TCEP is added. Or you can make a stock solution of TCEP and pH that.

Also, I kept the unfolded protein in the urea/TCEP solution overnight in the cold and then centrifuged the solution, before applying to the column.

Thank you for the suggestions. I actually added 5 mM DTT and the protein eluted off the column fine. I assume the protein may have been non specifically forming disulfide bonds while in its unfolded state causing it to aggregate, or simply fail to refold. I have not had a chance yet to check if the purified protein is active, but will update once I do.

Forgot to update this. Was able to get active protein with the method above, but most of the protein comes out of solution soon after elution off the HIS column so I have not been able to get enough protein for crystallographic studies yet. I am able to get enough protein for assay purposes however.